Deeply subwavelength near infrared imaging with avalanching nanoparticles - PROJECT ABSTRACT Optical microscopy is ubiquitous in cell biology and is well-suited for studying cell signaling in live cells and animals, offering minimally invasive in situ monitoring, subcellular and millisecond resolution, and probes for specific receptors or other biomolecules. However, live cell imaging is constrained by the diffraction limit, by temporal limits of most super-resolution techniques, by probe stability, and by the UV or visible lasers used in most microscopy. These common microscopy lasers can be phototoxic, give rise to autofluorescent background, photobleach fluorophores to generate harmful free radicals, and lack the ability to penetrate to subsurface sites. Here, we address all of these limitations with unique new optical probes, avalanching nanoparticles (ANPs), that show the most nonlinear emission of any reported nanoscale material. ANPs upconvert incident light, which enables them to be imaged without autofluorescent background or photobleaching, and to be excited by near infrared (NIR) wavelengths, which are far less phototoxic than the lasers most common in bioimaging. The steep nonlinearity of ANP emission enables experimental realization of a revised Abbe equation of the diffraction limit, achieving real-time sub-70 nm spatial resolution without the need for special optics, computation, or imaging processing. To enable live-cell ANP imaging, we propose synthesis of biocompatible and antibody-functionalized ANPs for receptor targeting, as well as construction of a modified laser scanning confocal microscope, which addresses the innate slow kinetics of photon avalanching. Both breast cancer cells and neural microglia will be imaged to determine spatial localization and dynamics of cell surface receptors critical to their respective functions. ANPs may also be photoswitched indefinitely between bright and dim states entirely with NIR light, enabling sub-nanometer localization accuracies with the superresolution technique INPALM. Because ANPs do not measurably photobleach, collection of unlimited photons with controlled NIR toggling between bright and dim states is possible, leading to this exceptional precision. While these optical breakthroughs have the potential to fundamentally alter how high-resolution bioimaging is approached, ANPs have not been imaged in live cells and only in limited cases in fixed cells. We propose imaging of microglial receptors in live cells at 70-nm resolution and INPALM imaging of fixed cells at sub-nanometer precision with a confocal microscope customized for ANP imaging. The combination of receptor-targetable ANPs with a microscope designed for fast, high-resolution ANP imaging and for ANP superresolution leads to the ultimate goal of bringing these radical advances in nanoparticle optics into the realm of bioimaging.
